RESUMEN
Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency <1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK--a nonreceptor tyrosine-kinase of the src family of proto-oncogenes--is expressed in beta-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of beta-cell function, the deficit of which may lead to the development of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Mutación , Familia-src Quinasas/genética , Adolescente , Adulto , Animales , Western Blotting , Línea Celular Tumoral , Análisis Mutacional de ADN , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Salud de la Familia , Femenino , Predisposición Genética a la Enfermedad , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/patología , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Microscopía Confocal , Persona de Mediana Edad , Linaje , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto Joven , Familia-src Quinasas/metabolismoRESUMEN
A novel frameshift mutation attributable to 14-nucleotide insertion in hepatocyte nuclear factor-1alpha (HNF-1alpha) encoding a truncated HNF-1alpha (G554fsX556) with 76-amino acid deletion at its carboxyl terminus was identified in a Thai family with maturity-onset diabetes of the young (MODY). The wild-type and mutant HNF-1alpha proteins were expressed by in vitro transcription and translation (TNT) assay and by transfection in HeLa cells. The wild-type and mutant HNF-1alpha could similarly bind to human glucose-transporter 2 (GLUT2) promoter examined by electrophoretic mobility shift assay (EMSA). However, the transactivation activities of mutant HNF-1alpha on human GLUT2 and rat L-type pyruvate kinase (L-PK) promoters in HeLa cells determined by luciferase reporter assay were reduced to approximately 55-60% of the wild-type protein. These results suggested that the functional defect of novel truncated HNF-1alpha (G554fsX556) on the transactivation of its target-gene promoters would account for the beta-cell dysfunction associated with the pathogenesis of MODY.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Transportador de Glucosa de Tipo 2/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Eliminación de Secuencia , Activación Transcripcional/genética , Animales , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , RatasRESUMEN
OBJECTIVE: Six known genes responsible for maturity-onset diabetes of the young (MODY) were analysed to evaluate the prevalence of their mutations in Thai patients with MODY and early-onset type 2 diabetes. PATIENTS AND METHODS: Fifty-one unrelated probands with early-onset type 2 diabetes, 21 of them fitted into classic MODY criteria, were analysed for nucleotide variations in promoters, exons, and exon-intron boundaries of six known MODY genes, including HNF-4alpha, GCK, HNF-1alpha, IPF-1, HNF-1beta, and NeuroD1/beta2, by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method followed by direct DNA sequencing. Missense mutations or mutations located in regulatory region, which were absent in 130 chromosomes of non-diabetic controls, were classified as potentially pathogenic mutations. RESULTS: We found that mutations of the six known MODY genes account for a small proportion of classic MODY (19%) and early-onset type 2 diabetes (10%) in Thais. Five of these mutations are novel including GCK R327H, HNF-1alpha P475L, HNF-1alphaG554fsX556, NeuroD1-1972 G > A and NeuroD1 A322N. Mutations of IPF-1 and HNF-1beta were not identified in the studied probands. CONCLUSIONS: Mutations of the six known MODY genes may not be a major cause of MODY and early-onset type 2 diabetes in Thais. Therefore, unidentified genes await discovery in a majority of Thai patients with MODY and early-onset type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Mutación , Adulto , Edad de Inicio , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diabetes Mellitus Tipo 2/epidemiología , Femenino , Quinasas del Centro Germinal , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/genética , Factor Nuclear 4 del Hepatocito , Proteínas de Homeodominio/genética , Humanos , Masculino , Linaje , Proteínas Serina-Treonina Quinasas/genética , Tailandia/epidemiología , Transactivadores/genética , Adulto JovenRESUMEN
A20 or tumor necrosis factor (TNF)-induced protein 3 (TNFAIP3) is a negative regulator of nuclear factor-kappaB (NF-kappaB). We have investigated whether polymorphisms in this gene are associated with increased atherosclerosis in diabetic patients. Five tag single nucleotide polymorphisms (SNPs) were typed in 479 type 2 diabetic patients from Boston, including 239 coronary artery disease (CAD)-positive case subjects and 240 CAD-negative control subjects. Two tag SNPs (rs5029930 and rs610604) were independently associated with CAD; adjusted odds ratios (ORs) for minor allele carriers were 2.3 (95% CI 1.4-3.8, P = 0.001) and 2.0 (1.3-2.9, P = 0.0008), respectively. The association with rs610604 was dependent on glycemic control, with ORs of 3.9 among subjects with A1C < or =7.0% and 1.2 for those with A1C >7.0% (P for interaction = 0.015). A similar interaction pattern was found among 231 CAD-positive and 332 CAD-negative type 2 diabetic patients from Italy (OR 2.2, P = 0.05 vs. OR 0.9, P = 0.63 in the low vs. high A1C strata, P for interaction = 0.05). Quantitative RT-PCR in blood mononuclear cells from 83 nondiabetic subjects showed that rs610604 and rs5029930 minor allele homozygotes have 30-45% lower levels of A20 mRNA than major allele homozygotes, and heterozygotes have intermediate levels (P = 0.04 and 0.028, respectively). These findings point to variability in the A20/TNFAIP3 gene as a modulator of CAD risk in type 2 diabetes. This effect is mediated by allelic differences in A20 expression.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Complicaciones de la Diabetes/genética , Diabetes Mellitus Tipo 2/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleótido Simple , Anciano , Alelos , Boston , Proteínas de Unión al ADN , Exones , Femenino , Regulación de la Expresión Génica , Genotipo , Homocigoto , Humanos , Italia , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Riesgo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
CONTEXT: Six maturity onset diabetes of the young (MODY) genes have been discovered to date but account for a small proportion of MODY among Asians, suggesting the existence of other MODY genes in this racial group. OBJECTIVE: The aim of this study was to investigate whether or not genetic variants in PAX4, a crucial transcription factor in beta-cell development, contribute to MODY in Thais. DESIGN AND METHODS: We screened PAX4 coding sequences in 46 MODY probands without mutation in known MODY genes and in 74 nondiabetic controls using PCR-single-stranded conformational polymorphism analysis followed by direct sequencing. Genotyping of variants identified was done by PCR-restriction fragment length polymorphism analysis. RESULTS: Eight sequence differences were identified. Two novel variations (R164W and IVS7-1G>A) were found in two different probands. Neither was found in the 74 nondiabetic controls and additional 270 healthy subjects of Thai origin. R164W segregated with diabetes in the family of the proband and in vitro studies showed that it impairs the repressor activity of PAX4 on the insulin and glucagon promoters. The remaining six variants were previously described and observed in both groups. One of them, R192H, was three times more frequent in MODY probands than in 342 nondiabetic controls (minor allele frequency = 0.196 vs. 0.064; P < 0.00001). The same variant was associated with a younger age at diagnosis among 254 Thai subjects with adult-onset type 2 diabetes (44.6 +/- 15 vs. 49.7 +/- 11 yr; P = 0.048). CONCLUSIONS: We have identified two possible pathogenic mutations of PAX4, R164W, and IVS7-1G>A. For one of these, we have shown evidence of segregation with diabetes and a functional impact on PAX4 activity. Single-nucleotide polymorphism R192H might influence the age at onset of diabetes.
Asunto(s)
Pueblo Asiatico/genética , Diabetes Mellitus Tipo 2/etnología , Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodominio/genética , Factores de Transcripción Paired Box/genética , Adulto , Edad de Inicio , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Linaje , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , TailandiaRESUMEN
In order to investigate whether there would be any association between abnormalities of either reg1 alpha or reg1 beta gene and diabetes mellitus in man, these two genes were analyzed in 42 patients with type 1 diabetes mellitus, 12 with fibrocalculous pancreatopathy, 37 with type 2 diabetes mellitus, and 22 normal controls, by PCR-SSCP analysis and nucleotide sequencing technique. Polymorphism in the reg1 alpha gene resulted in three mobility patterns in the PCR-SSCP analysis, due to nucleotide constituents at position -10 before exon 1 being either C/C, T/C or T/T. These three mobility patterns were observed in every group of subjects. The analysis of reg1 beta gene showed nucleotide substitutions in exon 4 in one patient, exon 5 in another patient with type 1 diabetes, and in exon 4 and intron 5 in one patient with fibrocalculous pancreatopathy. The nucleotide substitutions in exon 4 in the patient with type 1 diabetes and that with fibrocalculous pancreatopathy occurred at codons 103 and 84 while that in exon 5 in the patient with type 1 diabetes occurred at codon 141, changing the codons from CAT to CAC, GTG to GCG, and ACT to AAT and resulting in H103H silent, V84A and T141N missense mutations, respectively. In conclusion, using PCR-SSCP and nucleotide sequence analyses, we did not find any association between abnormalities of either reg1 alpha or reg1 beta gene with any type of diabetes studied.
Asunto(s)
Proteínas de Unión al Calcio/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Mutación , Proteínas del Tejido Nervioso , Adulto , Codón , Exones , Femenino , Humanos , Litostatina , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADNRESUMEN
We measured the cell-mediated immune response to GAD and bovine beta-casein in 38 type 1 and 37 type 2 diabetic patients who visited diabetic clinics or who were hospitalized in Bangkok, Thailand, and in 43 normal controls, by using a lymphoproliferation assay. Positive results against GAD were found in 29/38 (76.3%) type 1, 6/37 (16.2%) type 2 diabetic patients and 1/43 (2.3%) normal controls. Positive results against bovine beta-casein were found in 18/38 (47.4%), 5/37 (13.5%) and 1/43 (2.3%) of these subjects, respectively. The frequencies of the positive results and the magnitude of the responses to both antigens in type 1 diabetic patients were significantly higher than those in the other two groups (P<0.001). In addition, the prevalence of a positive lymphoproliferative response to these antigens in type 1 diabetic patients was higher than that of anti-GAD antibody positivity in the same group of subjects (26.3%). Thus, the prevalence of positive lymphoproliferative response to GAD in type 1 diabetic patients studied was higher than the prevalence of other autoimmune markers previously reported in type 1 diabetic patients in Thailand.
Asunto(s)
Autoanticuerpos/sangre , Caseínas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Glutamato Deshidrogenasa/inmunología , Animales , Bovinos , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Humanos , Inmunidad Celular , Activación de Linfocitos , Valores de Referencia , Análisis de Regresión , Reproducibilidad de los Resultados , TailandiaRESUMEN
Fibrocalculous pancreatopathy is a form of diabetes, associated with tropical chronic calcific pancreatitis, in which islet beta-cell loss and pancreatic stone formation are found. It is likely to be a multifactorial disease with both genetic and environmental components. Regenerating (reg) gene encodes protein that has been involved in pancreatic lithogenesis and the regeneration of islet cells and therefore the abnormality of reg genes could be associated with fibrocalculous pancreatopathy. In this study, regla and reg1beta mRNAs were isolated from peripheral blood lymphocytes obtained from 16 patients with fibrocalculous pancreatopathy, 42 patients with type 1 diabetes, 37 patients with type 2 diabetes, and 22 normal controls. mRNAs were amplified by reverse-transcription polymerase chain reaction (RT-PCR) and analysed by a single strand conformation polymorphism (SSCP) technique. The reg1alpha and reg1beta mRNAs were isolated, indicating the ectopic expression of these genes in peripheral blood lymphocytes; however, variation among mobility patterns was not observed in the SSCP analysis of the RT-PCR products. The results indicated that there was no abnormality of the regla and reg1beta mRNAs obtained from the study groups.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas del Tejido Nervioso , Enfermedades Pancreáticas/genética , ARN Mensajero/genética , Enzimas de Restricción del ADN/metabolismo , Electroforesis en Gel de Agar , Humanos , Litostatina , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , TailandiaRESUMEN
Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. We recently found that the detection of cell-mediated immune response to glutamic acid decarboxylase (GAD) was more useful than the detection of specific autoantibodies for the diagnosis of type 1 diabetes mellitus. In this study, we established a flow cytometric analysis for the detection of activated T cells in whole venous blood, obtained from diabetic patients and normal controls after stimulation by GAD. Two millitiers of peripheral venous blood and 6 hours incubation time were used for performing the test. It was found that 33% (3/9) type 1 diabetic patients, 7.7% (1/13) type 2 diabetic patients and neither patients with fibrocalculous pancreatopathy nor normal controls had > or = 20% CD8+ T cells expressing CD69. The results suggest that flow cytometry may be a useful tool for the detection of surrogate markers of type 1 diabetes mellitus.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Citometría de Flujo , Glutamato Descarboxilasa/biosíntesis , Glutamato Descarboxilasa/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Adolescente , Adulto , Anciano , Antígenos de Diferenciación de Linfocitos T/metabolismo , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inmunidad Celular/inmunología , Lectinas Tipo C , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , TailandiaRESUMEN
Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin-receptor (IR) signaling and, when over-expressed, induces insulin resistance in vitro and in vivo. Understanding the regulation of ENPP1 expression may, thus, unravel new molecular mechanisms of insulin resistance. Recent data point to a pivotal role of the ENPP1 3'UTR, in modulating ENPP1 mRNA stability and expression. We sought to identify trans-acting proteins binding the ENPP1-3'UTR and to investigate their role on ENPP1 expression and on IR signaling. By RNA electrophoresis mobility shift analysis and tandem mass spectrometry, we demonstrated the binding of heat shock protein 70 (HSP70) to ENPP1-3'UTR. Through this binding, HSP70 stabilizes ENPP1 mRNA and increases ENPP1 transcript and protein levels. This positive modulation of ENPP1 expression is paralleled by a reduced insulin-induced IR and IRS-1 phosphorylation. Taken together these data suggest that HSP70, by affecting ENPP1 expression, may be a novel mediator of altered insulin signaling.